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Calcium chloride dihydrate

氯化钙二水合物

Company: Sigma-Aldrich
Catalog#: C3306
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Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
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Date:
2018-01-20
[Abstract]  With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the extensively studied Gram-negative bacterium Escherichia coli, as it is considered as the workhorse for both research and industrial purposes. Here, we present a simple, robust and effective protocol using the CRISPR/Cas9 system in combination with the λ Red machinery for gene knockout in E. coli. Crucial in our procedure is the use of a double-stranded donor DNA and a curing strategy for removal of the guide RNA encoding plasmid ... [摘要]  随着CRISPR / Cas9技术作为基因组编辑的标准工具的最近实施,全世界的实验室正在经历PCR以来分子生物学方面最大的进步之一。这种方法的关键优点是其简单和普遍适用于任何物种的门。特别感兴趣的是广泛研究的革兰氏阴性细菌大肠杆菌,因为它被认为是研究和工业用途的主力。在这里,我们提出了一个简单,强大和有效的协议,使用CRISPR / Cas9系统结合λ红色基因敲除机器。大肠杆菌。在我们的程序中最重要的是使用双链供体DNA和固化策略来去除导向RNA编码质粒,其允许在仅仅两个工作日后开始新的突变。我们的方案允许多个具有高诱变效率的基因敲除株,适用于高通量的方法。

【背景】革兰氏阴性细菌大肠杆菌是生物技术工程中最重要的生物之一。已在能源,农业,食品生产,生物技术,医药等不同行业的各种流程中成功实施。由于不断的技术进步,生物技术部门正在迅速发展。特别是,CRISPR / Cas9技术可能是PCR(分子)生物学最大的革命(Ledford,2015)。简而言之,CRISPR / Cas9保护细菌免受诸如质粒和病毒等侵入性遗传因子的影响(Marraffini,2015)。利用这种从原核生物获得的免疫系统,已经开发了基于CRISPR / Cas9系统的基因组编辑的非常有力的工具(Jinek等人,2012)。

CRISPR / ...

Aldicarb-induced Paralysis Assay to Determine Defects in Synaptic Transmission in Caenorhabditis elegans
Author:
Date:
2017-07-20
[Abstract]  Aldicarb treatment causes an accumulation of acetylcholine in the synaptic cleft of the neuromuscular junction, resulting in sustained muscle activation and eventually paralysis. Aldicarb-induced paralysis assay is an easy and fast method to determine whether synaptic transmission of a C. elegans mutant of interest is altered. This assay is based on the correlation of the rate of neurotransmitter release with the rate of paralysis. In this protocol, we describe a method for simultaneously assessing the aldicarb sensitivity of animals with different genotypes. [摘要]  涕灭威的治疗导致神经肌肉接头突触裂缝中的乙酰胆碱积聚,导致持续的肌肉活化并最终导致麻痹。 涕灭威诱导的麻痹测定是确定是否突触传播的一种简单且快速的方法。 线虫兴趣突变体被改变。 该测定是基于神经递质释放速率与麻痹率的相关性。 在该方案中,我们描述了同时评估具有不同基因型的动物的涕灭威敏感性的方法。
【背景】突触传递通过动作电位到达突触前末端而开始,这又导致神经递质的释放。释放的神经递质结合并激活突触后受体(Sudhof,2013)。 ℃。线虫运动由乙酰胆碱释放兴奋性运动神经元和GABA(γ-氨基丁酸)释放抑制运动神经元控制(Richmond和Jorgensen,1999; ...

Quantification of Triphenyl-2H-tetrazoliumchloride Reduction Activity in Bacterial Cells
Author:
Date:
2017-01-20
[Abstract]  This protocol describes the use of the 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) salt to evaluate the cell redox potential of rhizobia cells. The production of brightly colored and insoluble 1,3,5-Triphenyltetrazolium formazan arising from TTC reduction is irreversible and can be easily quantified using a spectrophotometer. This protocol allows the production of reproducible results in a relatively short time for Sinorhizobium meliloti 1021 cells grown both in exponential and stationary phases. The results here presented show that the S. meliloti cells deriving from exponential-phase cultures had increased cell redox potential as compared to the ones deriving from stationary-phase cultures. This means that under exponential growth conditions the S. meliloti ... [摘要]  该方案描述了使用2,3,5-三苯基-2H-四唑氯化物(TTC)盐来评估根瘤细胞的细胞氧化还原电位。由TTC还原产生的鲜色不溶性1,3,5-三苯基四唑甲The的生产是不可逆的,可以使用分光光度计轻松地定量。该方案允许在相对较短的时间内在指数阶段和固定阶段生长的中华根瘤菌中华根瘤菌1021细胞产生可重复的结果。这里提出的结果表明,与来自固定相培养物的细胞相比,从指数阶段培养物得到的猕猴桃细胞具有增加的细胞氧化还原电位。这意味着在指数增长条件下, meliloti 细胞产生更大量的TTC减少所需的还原等同物。

背景 TTC盐是一种水溶性和无色的化合物,可以还原成甲an,高度着色的化合物。甲醛的不可逆形成可以使用分光光度计进行定量。由于其性质和其降低的潜力,该四唑盐广泛用于真核生物和原核生物两者,作为细胞氧化还原活性,活力,药物敏感性和底物利用测定的指标(Byth等人, 2001; Hayashi等人,2003; Raut等人,2008; Lin等人,2008)。由于膜电位,四唑鎓盐的净正电荷有助于细胞摄取,从而允许其细胞内还原(Berridge等人,2005)。在原核生物中,TTC降低的主要研究涉及革兰氏阴性呼吸细菌大肠杆菌,而对Rhizobiacea 家族的成员只有少数研究报道。在这个协议中,这个家族最好的遗传特征成员之一, ...

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